The type 2 iodothyronine deiodinase (D2) is a pivotal regulatory selenoenzyme catalyzing the first step in thyroid hormone action, the conversion of thyroxine (T4) to the active hormone, 3,5,3'-triiodothyronine (T3). Studies supported by this proposal have led to the novel conclusion that the activity of this enzyme is regulated post-translationally by a dynamic balance between ubiquitination, causing D2 inactivation and proteasomal degradation, and deubiquitination, which reactivates it. Our goals are to identify the proteins and regulatory steps in these two processes, and place this newly recognized mechanism for maintaining T3 homeostasis into a physiological context. D2 is an endoplasmic reticulum (ER) integral membrane protein selectively ubiquitinated and targeted to the proteasomal system, a process termed ER-associated degradation (ERAD). We identified the ERAD-related UBC6 and UBC7 as important ubiquitin conjugases (E2s) for D2. We hypothesize that D2 ubiquitination is initiated after recognition by an as yet unidentified D2-specific ubiquitin ligase (E3) that associates with these E2s. Specific Aim I will identify the critical E3s, their contact sites and their function. D2 is constantly ubiquitinated under basal conditions but this is accelerated by T4 catalysis. The identity of the critical steps in these processes is unknown. Is substrate-induced ubiquitination a similar, but faster, reaction than basal ubiquitination or are there different sets of E2s and D2-specific E3 involved? Since the process we are investigating is complex, Specific Aim II will use in vivo systems including mammalian two hybrid systems and peptides which can block E3 binding to confirm the in vitro results and define T4 related events. Using a yeast-two-hybrid system, we identified two closely related D2-binding ubiquitin-specific processing proteases (UBPs) that specifically deubiquitinate and reactivate D2. In brown adipose tissue, cold exposure induces a rapid increase one of these, activating ubiquitinated D2 and providing active enzyme more rapidly than does de novo synthesis. Little is known of these only recently identified UBPs and Specific Aim III will define their D2 interaction mechanism, developmental expression and regulation by T3, cAMP and other hormones. PUBLIC HEALTH RELEVANCE: Thyroid hormone is a critical hormone for all vertebrate development and control of energy expenditure in humans. This project is to understand new mechanisms involved in the regulation of thyroid hormone action. Once completed, we will be better equipped to develop pharmacological tools to regulate thyroid hormone action on a tissue-specific basis, which is extremely relevant for obesity, insulin resistance and the metabolic syndrome.